Anticoagulants are useful therapeutic agents in the pharmacological treatment of, for example, acute deep venous thrombosis, pulmonary embolism, acute arterial embolization of the extremities, myocardial infarction, and disseminated intravascular coagulation. Prophylactic administration of anticoagulants is believed to prevent a recurrence of embolism in patients with rheumatic or arteriosclerotic heart disease and to prevent certain thromboembolic complications of surgery. Administration of anticoagulants has also been indicated in the treatment of coronary artery and cerebrovascular disease. Arterial thrombosis, particularly in arteries supplying the heart muscle and brain, is a leading cause of death.
Hirudin is a 65 amino acid residue polypeptide isolated from the salivary glands of leeches. It is a thrombin specific inhibitor and therefore can be used as an anticoagulant agent. Although quite potent, clinical use of hirudin isolated from leech extracts seems unlikely because of its limited quantity, expense, and allergic reactions which commonly follow administration of any foreign protein of this size.
Originally, applicants discovered a specific region of hirudin that is responsible, at least in part, for its anticoagulant activity . The peptide region (amino acid residues 55 to 65 of hirudin) was chemically synthesized and shown to bind the recognition site of thrombin; the recognition site being spatially distinct from the the enzymatic cleavage site. Binding of synthetic peptides were also shown to competitively prevent binding of fibrinogen to the recognition site of thrombin, an important prerequisite to fibrin production and clot formation, and are thereby of potential medical value as anticoagulants.
Applicants have now prepared certain iodotyrosine, and radioisotopically labeled tyrosine derivatives of this peptide. The present invention provides a method for labeling hirudin peptides while preserving the binding site activity present. The iodotyrosine peptides and radio-isotope derivatives in this invention also maintain their parental attributes as anticoagulants, and thus may also allow for a scientifically interesting and therapeutically significant adjuncts. Specifically, the ability to incorporate radioactive isotopes of hydrogen and iodine into the peptide and maintain biological activity of the peptide provides a new important reagent in pharmaceutical testing and usage of anticoagulants. Such reagents would be expected to be important in animal and biochemical studies i.e., radioimmuneassays, screening of drug agonists and antagonists, pharmacokinetic and biodistribution studies, and diagnostic imaging. Moreover, the presence of the iodotyrosine or radio-isotopes thereof, may prove of value in drug development and testing of antithrombotic agents and such derivatives may in themselves have enhanced potency and extended duration of action.